1. Field of the Invention
The present invention relates to plasmid cloning vectors having a site, derived from transposon Tn904, which is cleaved by the restriction endonuclease AvaI and which encodes for streptomycin resistance and to a method for producing the cloning vectors. The present invention particularly relates to cloning vectors including the AvaI site from Tn904 in a parent vector derived from pRO1600 and pBR322.
2. Prior Art
Many transposons have been described for procaryote microorganisms. These transposons may be divided into two groups based on the composition of their distal regions. One such group, typified by Tn3, was obtained from the R-plasmid, R1. The distal regions of its DNA are sequences comprised of 38 base pairs which occur in reverse orientation (Ohtsubo, H., et al. Cold Spring Harbor Symp. Quant. Biol. 43:1269-1277 (1979)). These base pairs are absolutely required for transposition and mutations in these regions cannot be complemented in trans for transposition to occur (Gill, R. et al. Nature 282:801-806 1979; Heffron, F. et al. Proc. Nat. Acad. Sci. USA 74:702-706 (1977); Heffron, F., et al. Cell 18:1153-1163 (1979); Heffron, F., et al. Proc. Nat. Acad. (1979)). This organization is in marked contrast to Tn5, a transposon encoding Kanamycin resistance. The distal regions of Tn5 are characterized by DNA sequences in reverse orientation of 1.5 kilobase pairs each and the region which encodes Kanamycin resistance extends internally from one of the distal repeated sequences to the central, non-repeated regions of the transposon (Jorgenson R. A., et al. Mole, Gen. Genet. 177:65-72 (1979); Rothstein, S. J., et al. Cell 19:795-805 (1980); Rothstein, S. J. et al. Cold Spring Harbor Symp. Quant. Biol. 45:99-105 (1980)). The genes associated with transposition have been shown to be encoded by the other reverse repeat sequence (Auerswald, E. et al. Cold Spring Harbor Symp. Quant. Biol. 45:107-113 (1980); Rothstein, S. J., et al. Cell 19:795-805 (1980); Rothstein R. J., et al. Cold Spring Harbor Symp. Quant. Biol. 45:99-105 (1980)). In addition, it has been shown that either of the inverted repeats of Tn5 can transpose independently of the rest of the transposon (Berg, D. E., et al. Cold Spring Harbor Symp. Quant. Biol. 45:115-123 (1980)). Thus, two groups of transposons differing significantly in the size and function of their distal regions have been observed although there remains, also, significant diversity of structure and function within either of the two groups.
Many R-plasmids exist as a single replicon while other R-plasmids have been shown to exist as separate components, one bearing the R-determinants specifying resistance to antibiotics and the other encoding transfer functions. These multi-component R-plasmids are called aggregates and another example of an aggregate R-plasmid would be the combination of a transferable and a non-transferable plasmid aggregate wherein both plasmids would transfer together in bacterial matings selecting for the transfer of the transfer-defective plasmid, Ohtsubo, H., et al. Cold Spring Harbor Symp. Quant. Biol. 43:1269-1277 (1979).
The mobilization of pRO161, a non-transferable plasmid, when Tn1 transposed from pRO161 to a sex factor maintained by the same host bacterium has been described, Ohtsubo, H., et al. Cold Spring Harbor Symp. Quant. Biol. 43:1269-1277 (1979); Olsen, R. H., et al. J. Bacteriol. 113:772-780 (1973). This activity of Tn1 may have been associated with the formation of Tn1-mediated cointegrates during conjugation which resolved into separate plasmids in the newly-formed transconjugants, Campbell, A. D., et al. pp15-22. In: A. I. Bukhari et al. Cold Spring Harbor Laboratories, Cold Spring Harbor, New York (1977); Hansen, J. B., et al., J. Bacteriol. 135:227-238 (1978); Royle, P., et al., J. Bacteriol. 145:145-155 (1981). Transposition of Tn1 from pRO161 to either Pseudomonas aeruginosa sex factor, FP2, or the R-plasmid, R388, subsequently facilitated mobilization of pRO161 by FP2::Tn1 (e.g., pRO271) or R388::Tn1 (e.g., pRO231). Thus, plasmid mobilization ability (Pma) is presumably due to trans-diploid homology between the transfer proficient and defective components of the plasmid aggregate that occurs when both plasmids have a copy of Tn1.
Transposon-mediated plasmid donor ability (Pma) of a nonconjugal plasmid, has been reported for pRO161, Ohtsubo, H., et al. Cold Spring Harbor Symp. Quant. Biol. 43:1269-1277 (1979). The association of Pma with the transposition of Tn1 from pRO161 to the P. aeruginosa sex factor, FP2; the resulting FP2::Tn1 mobilized pRO161 was shown. In these reports, transconjugants from matings were described whose phenotype resembles the result of analogous mobilizations using different plasmids as reported by Crisona et al., Cold Spring Harbor Symp. Quant. Biol. 45:115-123 (1980). These phenotypes included the occurrence of unstable cointegrate plasmids formed from the sex factor and the Tra.sup.- plasmid of the aggregate donor as well as the dissociation products of the cointegrate, i.e., sex factor and the Tra.sup.- plasmids. Our previous results, then, with Tn1-mediated Pma, resembled those of Crisona et al. with Tn3, J. Bacteriol. 142:701-713 (1980).
A recent report of Van Gijsegem and Toussaint described the formation of R-prime plasmids in Enterobacteriaciae which is facilitated by the transposable element, mini-Mu, Plasmid 7:30-44 (1982). These authors showed the formation of RP4-primes which include hetero-DNA (derived in this case from the chromosome) whose termini are defined by mini-Mu in direct repeat orientation. This structure may be analagous to the cointegrate intermediate proposed by the prior art for transposon-mediated Pma. (Olsen, R. H. J. Bacteriol. 133:210-216 (1978); Olsen, R. H., et al. J. Bacteriol. 125:837-844 (1976); Crisona, N. J., et al. J. Bacteriol. 142:701-713 (1980)). It has been observed that plasmids of the IncP-1 and IncP-2 groups formed recombinants in a recombination deficient (recA) host (Jacoby, G. A., et al. J. Bacteriol. 127:1278-1285 (1976)). One of the examples they cited was a recombination between plasmids RP1 and pMG1 resulting in the acquisition of Sm.sup.r by RP1. They suggested that recombination occurred reflecting homologous insertion sequences on IncP-1. Tn904, the Sm.sup.r -transposon described in this application has been useful for labeling the octopine-Ti plasmid of Agrobacterium tumefacinens (Klapwijk, P. M. et al. J. Bacteriol. 141:129-136 (1980)). However, Ooms et al. Cold Spring Harbor Symp. Quant. Biol. 45:99-105 (1980) found that the acquisition of Tn904 resulted in variable increases in the size of the target Ti plasmid. These authors suggested that the variable increase in size of the Ti plasmid subsequent to the addition of Tn904 might reflect amplification of the DNA sequence encoding Sm.sup.r during selection for high levels of streptomycin resistance (2 mg/ml).
The prior art has not constructed plasmid cloning vectors using Tn904. Such vectors would be very useful for allowing DNA to be inserted in the AvaI site with certainty that it was located in the site.